Infertility is a problem that plagues many couples. There are a great number of reasons why difficulty in achieving conception may be encountered, and, because of the complexity of the issues involved, this problem is still very prevalent. In recent years, our knowledge and understanding of the biology and chemistry of fertilization has expanded rapidly. Much of what we know is drawn from in vitro experiments with mouse gametes. It is believed that the strategem described for fertilization of mouse gametes applies to in vivo fertilization of most mammals, including humans.
It is well established that one of the major factors in reproductive failure can be the inability of the sperm to properly associate with, and penetrate, the egg. This type of infertility can be identified by the in vitro sperm binding/penetration assay utilizing the zona pellucida-free hamster egg. While the sperm of some individuals demonstrates a complete inability to penetrate the hamster egg, often there is also an accompanying significant reduction in sperm binding capacity (Kretzer, P., E. Pope, J. B. Younger, R. E. Blackwell [1987] "Long term follow-up of patients with zero hamster tests," The American Fertility Society [Abstract]).
The path leading to fertilization consists of several steps that occur in a precise order. Fertilization begins when sperm associates with ovulated eggs at the cell surfaces. The egg is surrounded by a thick extracellular matrix known as the zona pellucida. Attachment of sperm to the egg appears to be mediated in part by a specific zona pellucida glycoprotein ZP3 (Bleil, J. D., and P. M. Wasserman [1984] Dev. Biol. 104:243-347). The functional group of the glycoprotein is the oligosaccharide moiety of the molecule (Florman, H. M., K. B. Bechtl, and P. M. Wasserman [1984] Dev. Biol. 104:243-247; Shur, B. D. and G. Hall [1982] J. Cell Biol. 95:574). Both sperm adhesion and penetration following attachment are mediated by sperm associated proteins.
Several different sperm proteins are presently under investigation as candidates for the role of mediating egg oligosaccharide binding. All of these proteins are associated with the plasma membrane surrounding the sperm head. Evidence put forward by Shur and co-workers (Shur, B. D., and G. Hall [1982] Cell Biol. 102:1363-1372; Lopez, L. C., E. M. Bayna, D. Zitioff, N. L. Shaper, J. H. Shaper, and B. D. Shur [1985] J. Cell Biol. 101:1501-1510; Shur, B. D., and D. Bennett [1975] Develop. Biol. 21:243-259) suggests that the glycosyltransferase, .beta.1-4 galactosyltransferase, may in part be responsible for mediating fertilization of the egg by binding N-acetylglucosamine residues in the zona pellucida. Furthermore, sperm capacitation is associated with the release of specific sperm bound galactosyltransferase (GalTase) substrate. The surface transferase is thus exposed for interaction with egg cell-surface glycoproteins during fertilization. In addition, prior galactosylation of the zona pellucida glycoproteins by exogenous galactosyltransferase and UDP-galactose destroys sperm ability to bind to egg (Bayna, E. M., R. B. Runyan, N. F. Scully, J. Reichner, L. E. Lopez, and B. D. Shur [1986] Molec. Cellul. Biochem. 72:141-151).
.beta.1-4 galactosyltransferase is the most extensively studied of the galactosyltransferases involved in glycoprotein biosynthesis. This enzyme is involved in the addition of O- and N-linked glycoprotein oligosaccharides. Membrane associated galactosyltransferase has classically been localized to the Golgi-apparatus, but a portion of the cellular activity has been found at the cell surface. Numerous reports have suggested that cell-surface galactosyltransferase might be involved in many cellular functions including: cell adhesion (Roth, S., D. J. McGuire, and S. Roseman [1971] J. Cell. Biol. 51:536-547; Pierce, M., E. A. Turley, and S. Roth [1980] In: International Review of Cytology [Bourne, G. H. J. F. Danierl and K. W. Jeon, eds.] pp. 2-44, Academic Press, Orlando, Fla.), recognition (Roseman, S. [1970] Chem. Phys. Lipids 5:270-274), differentiation (Weiser, M. M. [1973] J. Biol. Chem. 248:2536-2549), embryogenesis (Shur, B. D. [1982] In: The Glycoconjugates Vol. 3, pp. 146-185, Academic Press, New York, N.Y.), and fertilization (Shur and Bennett [1975] supra; Bayna et al. [1986] supra).
Although different forms of galactosyltransferase have been characterized according to their localization within the cell, it has not been resolved as to how these different forms are generated. Recent studies suggest that the form of the enzyme may depend on translational differences due to alternate forms of mRNA (Shaper, J. H., G. F. Hollis, and N. L. Shaper [1988] Adv. in Second Messenger and Phosphoproteins Res. 322:39-42) or may depend on the level of phosphorylation of the enzyme (Strous, G. J., P. Van Kerkhof, R. J. Fallon, and A. L. Schwartz [1987] Eur. J. Biochem. 169:301-311.)
Human cDNA which was originally thought to be the clone for the .beta.1-4 galactosyltransferase gene (Humphreys-Beher, M. G., B. Bunnell, P. van Tiennen, D. L. Ledbetter, and V. J. Kidd [1986] Proc. Natl. Acad. Sci. USA 83:8918-8921) has now been identified as the cDNA of the gene encoding a protein that regulates galactosyltransferase (Bunnell, B. A., D. E. Adams, V. J. Kidd [1990] Biochem. Biophys. Res. Comm 171:196-203; Bunnell, B. A., L. S. Heath, D. E. Adams, J. M. Lahti, V. J. Kidd [1990] Proc. Natl. Acad. Sci. USA 87:7467-7471). This protein has been characterized as a Ca.sup.2+ /calmodulin dependent protein kinase which mediates phosphorylation and influences the activity of surface galactosyltransferase. The protein kinase has been termed galactosyltransferase activator, or GTA.